Cross-linking of smooth muscle caldesmon to the NH2-terminal region of skeletal F-actin.
The cross-linking of the F-actin-caldesmon complex with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide in the presence of N-hydroxysuccinimide generated four major adducts which were identified on polyacrylamide gels. By cross-linking 3H-actin to 14C-caldesmon, these were found to represent 1:1 cross-linked complexes of actin and caldesmon displaying different electrophoretic mobilities. Tropomyosin did not noticeably affect the cross-linking process. The same four fluorescent species resulting from the cross-linking of caldesmon to F-actin labeled with N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide were subjected separately to partial cleavages with hydroxylamine or cyanogen bromide. These treatments yielded fluorescent 41- and 37-kDa fragments, respectively, from each cross-linked entity indicating unambiguously that caldesmon was cross-linked only to the NH2-terminal actin stretch of residues 1-12. This region is also known to serve for the carbodiimide-mediated cross-linking of the myosin subfragment-1 heavy chain (Sutoh, K. (1982) Biochemistry 21, 3654-3661). A covalent caldesmon-F-actin conjugate containing a protein molar ratio close to 1:19 was isolated following dissociation of uncross-linked caldesmon. It showed a low level of activation of the ATPase activity of skeletal myosin subfragment-1, and the binding of Ca2(+)-calmodulin to the derivative did not cause the reversal of the ATPase inhibition. In contrast, the reversible binding of caldesmon to F-actin cross-linked to myosin subfragment-1 did not inhibit the accelerated ATPase of the complex. The overall data point to the dual involvement of the actin's NH2 terminus in the inhibitory binding of caldesmon and in actomyosin interactions in the presence of ATP.
Bartegi
A
A
Institut National de la Sante et de la Recherche Médicale U.249 Université de Montpellier I, France.
Fattoum
A
A
Kassab
R
R
D000199:Actins / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002234:Carbodiimides / Q000378:metabolism*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000378:metabolism*; D000070559:Gizzard, Non-avian / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism; D015879:Myosin Subfragments / Q000378:metabolism; D010449:Peptide Mapping; D011817:Rabbits; D014335:Tropomyosin / Q000378:metabolism
D000199:Actins; D002148:Calmodulin-Binding Proteins; D002234:Carbodiimides; D015879:Myosin Subfragments; D014335:Tropomyosin; D005022:Ethyldimethylaminopropyl Carbodiimide